Targeted Inactivation ofFrancisella tularensisGenes by Group II Introns

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Abstract
Studies of the molecular mechanisms of pathogenesis ofFrancisella tularensis, the causative agent of tularemia, have been hampered by a lack of genetic techniques for rapid targeted gene disruption in the most virulent subspecies. Here we describe efficient targeted gene disruption inF. tularensisutilizing mobile group II introns (targetrons) specifically optimized forF. tularensis. Utilizing a targetron targeted toblaB, which encodes ampicillin resistance, we showed that the system works at high efficiency in three different subspecies:F. tularensissubsp.tularensis, F. tularensissubsp.holarctica, and “F. tularensissubsp.novicida.” A targetron was also utilized to inactivateF. tularensissubsp.holarctica iglC, a gene required for virulence. TheiglCgene is located within theFrancisellapathogenicity island (FPI), which has been duplicated in the most virulent subspecies. Importantly, theiglCtargetron targeted both copies simultaneously, resulting in a strain mutated in bothiglCgenes in a single step. This system will help illuminate the contributions of specific genes, and especially those within the FPI, to the pathogenesis of this poorly studied organism.